pET28a-CohIII-CBM-HIS-ybbR
(Plasmid #60866)

• Purpose: Characteristic fingerprint molecule fused to cohesin type III domain to perform single molecule force spectroscopy experiments; covalently immobilized via introduced ybbR tag
• Depositing Lab: Hermann Gaub
• Publication: Schoeler et al Nat Commun. 2014 Dec 8;5:5635. doi: 10.1038/ncomms6635.

Backbone

• Vector backbone: pET28a
• Backbone manufacturer: Novagen
• Backbone size w/o insert (bp): 5216
• Total vector size (bp): 6527
• Vector type: Bacterial Expression

Growth in Bacteria

• Bacterial Resistance(s): Kanamycin, 50 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): DH5alpha
• Copy number: Low Copy

Gene/Insert 1

• Gene/Insert name: CohIII
• Alt name: cohesin type III
• Species: Ruminococcus flavefaciens
• Insert Size (bp): 615
• Promoter: T7

Cloning Information for Gene/Insert 1

• Cloning method: Gibson Cloning
• 5′ sequencing primer: T7 Forward
• 3′ sequencing primer: T7 Reverse

Gene/Insert 2

• Gene/Insert name: CBM
• Alt name: carbohydrate binding module
• Species: Clostridium thermocellum
• Insert Size (bp): 696
• Mutation: Cysteine 63 to Serine in CBM module
• Promoter: T7
• Tags / Fusion Proteins:
  • 6xHIS (C terminal on insert)
  • ybbR (C terminal on insert)
  • HRV 3C (C terminal on backbone)

Cloning Information for Gene/Insert 2

• Cloning method: Gibson Cloning
• 5′ sequencing primer: T7 Forward
• 3′ sequencing primer: T7 Reverse

Resource Information

• A portion of this plasmid was derived from a plasmid made by: We thankfully received the cohesin and CBM domain from Professor Edward Bayer (Department of Biological Chemistry, Weizmann Institute of Science, Israel) as recently described in this paper: "Atypical cohesin-dockerin complex responsible for cell surface attachment of cellulosomal components: binding fidelity, promiscuity, and structural buttresses."Salama-Alber Orly, Jobby Maroor K., Chitayat Seth, Smith Steven P., White Bryan A., Shimon Linda J. W., Lamed Raphael, Frolow Felix, Bayer Edward A. (The Journal of biological chemistry, 2013, 288 (23).

Terms and Licenses

• Academic/Nonprofit Terms:
  • UBMTA
• Industry Terms:
  • Not Available to Industry